Cd3 percp cy5.5 145 2c11

Unspecific binding was blocked by incubating cells with Fc receptor-blocking monoclonal antibody (clone 2.4G2; BD Biosciences) for 10 min. Cells were then stained with the following antibodies specifically binding: CD3-PerCP/Cy5.5 (145-2C11; BioLegend), CD4-BV785 (RM4-5; BioLegend), CD8-BV421 (53-6.7; BioLegend), B220/CD45R-APC-Cy7 (RA3-6B2; BioLegend), CD138-BV605 (281-2; BioLegend), CD69-PE (H1.2 F3; BioLegend), and PD-1-PE-Cy7 (29 F.1A12; BioLegend). Dead cells were excluded by ZombieGreen™ (BioLegend) staining, and doublets by scatter analysis. Cells were measured on a LSRII flow cytometer (BD) and analyzed by FlowJo software. In most samples, a minimum of 1 × 10⁵ events were measured.

Total blood cells or leukocytes from the liver or spleen were incubated with Fc block (eBiociences, Thermo Fisher Scientific) for 10 minutes at 4-8°C. Surface staining was performed for 20 minutes at 4-8°C and always included Fixable Viability Dye (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for exclusion of dead cells. The following conjugated anti-mouse mAbs (and respective clones) were used: CD3 PerCP-Cy5.5 (145-2C11), CD4 APC or Brilliant Violet (BV) 510 (GK1.5), CD8 BV711 (53-6.7), CD62L FITC (MEL-14), CD19 PE (1D3), CD25 APC (3C7), CD44 PE-Cy7 (IM7), CD45 Alexa Fluor 700 (30-F11), CD69 BV650 (H1.2F3), CD127 PE-Dazzle 594 (A7R34), NK1.1 PE-Cy5 (PK136), KLRG1 PE (MAFA), CXCR3 APC (CXCR3-173), TCR γδ BV421 (GL3), all from either BioLegend (San Diego, CA, USA) or Sysmex (Kōbe, Hyōgo, Japan). Cells were acquired in a BD LSR Fortessa X-20 cytometer and analyses were performed within live, single (based on FSC-A vs. FSC-W parameters) CD45+ leukocytes using FlowJo v10 (FlowJo, BD). For the clustering and visualization of high-dimensional data, equivalent numbers of live, single CD45+ cells from each condition were concatenated. Clustering was performed using X-shift (number of clusters determined by the algorithm) and data are presented using the dimensionality reduction method TriMAP (large-scale dimensionality reduction using triplets).